DNA was removed from each RNA preparation using Turbo DNA-free Kit (Ambion), according to manufacturer’s instructions. RNA quantity (A260) and purity (A260/280 ratio) were measured
in a NanoDrop 1000 Spectrophotometer https://www.selleckchem.com/products/XL880(GSK1363089,EXEL-2880).html (Thermo Fisher Scientific). cDNA was synthesised from 500 ng RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in a 20 μl reaction according to manufacturer’s protocols. Five μl of a 1:100 dilution of the cDNA reaction was used as template for qPCR amplification in 25 μl final volumes containing 12.5 μl of Power SYBR Green PCR Master Mix (Applied Biosystems) and 200 nM of each primer. Primers used for qPCR are listed in Table 2. The amplification was performed using StepOne PCR software (Applied Biosystems) with thermal cycling conditions set at 10 min at 95°C, followed by 40 Selleckchem Salubrinal cycles of 15 s at 95°C and 1 min at
60°C. Fluorescence was monitored during each extension phase and a melting curve analysis was performed after each run to confirm the amplification of specific transcripts. Each qPCR of the RNA samples was performed Veliparib in triplicate, no template was added in negative controls, and rpoB was used as internal control. The qPCR analysis was performed on three independent biological replicates. Slopes of the standard curves and PCR efficiency (E) for each primer pair were estimated by amplifying serial dilutions of the cDNA template. For quantification of mRNA transcript levels, Ct (threshold cycle) values of the target genes (gerAA) and the internal control gene (rpoB) derived from the same sample in each real-time PCR reaction were first transformed using the term E-Ct. The expression levels of target genes were then normalized by dividing their transformed Ct-values by the corresponding values obtained for internal control gene [64, 65]. Germination assays Storage water was decanted and the spores were resuspended in autoclaved Milli-Q water (20°C) immediately before heat activation at 65°C in a heating block (QBD2, Grant Instruments Ltd) for 20 min. The Morin Hydrate heat-activated
spores were rapidly cooled down by centrifugation for 3 min 4500 × g at 4°C before resuspension in germination buffer (200 mM K-phosphate buffer pH 7.2). The A600 of the buffered spore suspension was adjusted to ~2.1 (Shimadzu UV- 160A, Shimdazu Europe GMBH). L-Alanine (Sigma) was dissolved in Milli-Q water and filter sterilized prior to use through a 0.45 μm pore size filter. 100 μL of 0.05 – 0.2 M L-Alanine germinant solution was added to 100 μL buffered spore suspension in a 96-well microplate (BD) giving an initial A600 of ~1. Germination was by monitored by reading the drop in absorbance (A600) in a 96-well microplate reader (Tecan Infinite M200). Readings were performed at regular intervals (2 min) and the plate was shaken 10 s prior to each reading.